Compositions against candida infections

ABSTRACT

Provided herein are compositions for topical use comprising an imidazole or a salt thereof, and domiphen bromide as active ingredients for use in treating or preventing a fungal infection, wherein said imidazole is chosen from ketoconazole and clotrimazole as active ingredients for use in treating or preventing a fungal infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is filed as a continuation of U.S. application Ser. No.16/631,878 filed on Jan. 17, 2020, which is a national stage entry ofPCT/EP2018/068192 filed on Jul. 15, 2018, which claims priority to GBapplication 1711512.2 filed Jul. 18, 2018, and LU application LU100445filed Sep. 20, 2017, the contents of each of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates pharmaceutical compositions of miconazoleand quaternary ammonium salts against Candida infections, in particularrecurrent vaginal candidiasis.

ABSTRACT Mucosal biofilm-related fungal infections are very common andthe incidence of recurrent oral and vulvovaginal candidiasis issignificant.

Various antifungal compounds have been described to combat Candidainfections. A well-known composition for treatment of vulvovaginalcandidiasis comprises miconazole nitrate as active ingredient and itssold under de tradename Gyno Daktarin. The composition comprisespolyethylene glycol (PEG-6 and PEG-32) glycol stearate, oleoyl macrogolglycerides, liquid paraffin, butylhydroxyanisole, benzoic acid.

EP0832649 describes an synergistic effect of miconazoles and quaternaryammonium salts on cultures of Candida albicans, Staphylococcus aureusand a number of Trichopython species. US4091090 discloses topicalantifungal effect of domiphen bromide. Recurrent infections with Candidaare increasing and alternative more effective pharmaceuticalcompositions are desired.

SUMMARY OF THE INVENTION

The present invention identified novel combinations of miconazole (MC)and the quaternary ammonium salt domiphen bromide which is particularlyeffective against Candida sp. and this as well on in vitro cultures asin an experimental setting using a rat model with a vaginal candidainfection.

The present invention illustrates that domiphen bromide and miconazoledisplay synergistic antibiofilm activity, based on determination of thefractional inhibitory concentration indices. Using real-time propidiumiodide staining, domiphen bromide combined with miconazole , results inthe killing of biofilm cells already after 2 h treatment and providesa >3 LOG units reduction of cfu (colony forming units) in the treatedbiofilms after 24 h.

The invention is summarised in the following statements:

-   -   1. A composition for topical use comprising miconazole or a salt        thereof and domiphen bromide as active ingredients for use in        treating or preventing a fungal infection.    -   2. The composition according to statement 1, for use in treating        or preventing a fungal infection wherein the fungal infection is        an infection with Candida sp.    -   3. The composition according to statement 1 or 2, for use in        treating or preventing a fungal infection, wherein the fungal        infection is an infection with Candida albicans or Candida        glabrata.    -   4. The composition according to any one of statements 1 to 3,        for use in treating a biofilm formed by Candida sp.    -   5. The composition according to any one of statements 1 to 4,        for use in treating or preventing a vulvovaginal infection.    -   6. The composition according to any one of statements 1 to 5,        for use in treating or preventing a fungal infection, which is        formulated as a cream.    -   7. The composition according to any one of statements 1 to 6,        for use in treating or preventing a fungal infection, further        comprising a mucoadhesive.    -   8. The composition according to any one of statements 1 to 7,        for use in treating or preventing a fungal infection, wherein        the molar excess of miconazole or a salt over domiphen bromide        is between 2 and 4.    -   9. The composition according to any one of statements 1 to 8,        for use in treating or preventing a fungal infection wherein the        concentration of miconazole and its salt in said composition is        between 2 and 5% (w/w).    -   10. The composition according to any one of statements 1 to 9,        for use in treating or preventing a fungal infection, wherein        the molar excess of miconazole or a salt over domiphen bromide        is between 2 and 4.    -   11. The composition according to any one of statements 1 to 10,        for use in treating or preventing a fungal infection wherein the        concentration of miconazole nitrate is about 2% (w/w) and the        molar excess of miconazole nitrate over domiphen bromide is        about 3 .    -   12. The composition according to any one of statements 1 to 11,        for use in treating or preventing a fungal infection, wherein        the composition is applied over a period of 14 days.    -   13. A pharmaceutical composition comprising an physiologically        acceptable carrier and comprising as active antifungal        ingredients a mixture of miconazole or a salt thereof and        domiphen bromide.    -   14. The composition according to statement 13, comprising        miconazole nitrate.    -   15. The composition according to statement 13 or 14, which is a        crème with a viscosity of between 300 cp and 300 cp at 180 rmp.    -   16. The composition according to any one of statements 12 to 15,        which has a pH of between 2,7 and 3,5.    -   17. The composition according to statement any one of statements        12 to 16, wherein the molar excess of miconazole or a salt over        domiphen bromide is between 2 and 4.    -   18. The composition according to any one of statements 12 to 17,        wherein the concentration of miconazole and its salt in said        composition is between 2 and 5% (w/w).    -   19. The composition according to any one of statements 12 to 18,        further comprising a mucoadhesive.    -   20. The composition according to any one of statements 12 to 19,        which is packaged in an applicator for vaginal delivery.    -   21. A method treating or preventing a fungal infection        comprising the step of administering an effective amount of        miconazole or a salt thereof and domiphen bromide.    -   22. The method according to statement 21, wherein the fungal        infection is an infection with Candida sp.    -   23. The method according to statement 21 or 21, wherein the        fungal infection is a vulvovaginal infection.

DETAILED DESCRIPTION OF THE INVENTION

BRIEF DESCRIPTIONS OF THE FIGURES

Abbreviation used in the figures are: MCZ: miconazole; DM: domiphenbromide; BKC: benzalkonium chloride; DC: dequalinium bromide; TB;tetraethylammonium bromide; BTC: bezethonium chloride.

FIG. 1 . Combined domiphen bromide-miconazole treatment reducesmetabolic activity of C. albicans biofilm cells. The values representthe mean metabolic activity of 4 independent biological replicates,determined by CTB viability staining.

FIG. 2 . Metabolic activity of C. albicans biofilm cells aftercombination treatment of miconazole with quaternary ammonium compounds.The values represent the mean metabolic activity of 4 independentbiological replicates, determined by CTB viability staining.

2A: miconazole (MCZ) and benzalkonium chloride (BKC)

2B: miconazole (MCZ) and tetraethylammonium bromide (TB)

2C: miconazole (MCZ) and dequalinium chloride (DC)

FIG. 3 . PI fluorescence in C. albicans biofilms cells after combinationtreatment with miconazole and quaternary ammonium compounds. PIfluorescence was measured each 15 minutes during 17 h in C. albicansbiofilm cells after treatment with miconazole and domiphen bromide,benzalkonium chloride, bezethonium chloride or dequalinium chloride.

3A : miconazole and benzalkonium chloride

3B : miconazole and domiphen bromide

3C : miconazole and bezethonium chloride

3D : miconazole and dequalinium chloride

FIG. 4 . Survival of C. albicans biofilms cells after combinationtreatment with miconazole and quaternary ammonium compounds. Mean logCFU values ±SD are shown for at least 4 biological replicates.Statistical analysis was performed to assess significant differencesbetween single treatment with either tetraethylammonium bromide (50 μM),bezethonium chloride (50 μM), dequalinium chloride (12.5 μM),benzalkonium chloride (50 μM) and domiphen bromide (50 μM) orcombinations with miconazole. A 2-way ANOVA and Sidak's multiplecomparisons test was applied and significant P-values are shown.

FIG. 5 . Intravaginal fungal burden of all groups at different timepoints p.i.

The burden is shown as the log of the colony forming units (cfu) presenton the vaginal swab. Errors bars represent SEM. G: treatment group.

FIG. 6 : Intravaginal fungal burden of all groups.

The burden is shown as the area under the curve (AUC) compared to thecontrol group (G1) (=100%). shown as the log of the colony forming units(cfu) present on the vaginal swab. Errors bars represent SEM. G:treatment group.

FIG. 7 shows intravaginal fungal burden of all groups at different daysp.i.

FIG. 8 shows intravaginal fungal burden of all groups. The burden isshown as the area under the curve (AUC).

FIG. 9 summarises the result of intravaginal burden of differentformulations on day 4, 9 and 14 post infection.

FIG. 10 shows survival of C. glabrata biofilm cells after single -orcombination treatment with MCZ and DB.

FIG. 11 shows survival of C. albicans biofilm cells after single -orcombination treatment with an antimycotic and domiphen bromide.

FIG. 12 shows that combined MCZ-DB treatment reduces the MIC value ascompared to mono treatment of MCZ against C. glabrata planktoniccultures.

DEFINITIONS

About is used to described a deviation of 10% below and a above acertain value. Thus for example “about 10” means between 9 and 11,“about 50” means between 45 and 55.

Miconazole (((RS)-1-[2-(2,4-dichlorofenyl)-2-[(2,4-dichlorofenyl)methoxy] ethyl] imidazole) is an antifungal compound which interfereswith the metabolism of ergosterol, a component of the yeast cellmembrane. The compound is typically formulated as its nitrate.

Domiphen bromide [N,N-dimetyl-N-(2-phenoxyetyl)-, bromide] is aquaternary ammonium salt with antiseptic properties.

Viscosity in the context of the present invention refers to resistanceto gradual deformation by shear stress. Viscosity can be measured bye.g. a Brookfield HA viscometer.

Fungal infection refers to a disease caused by one or more selected fromthe list comprising Candida spp., Aspergillus spp., Cryptococcus spp.,Pneumocystis spp., Zygomyces spp., Dermatophytes, Blastomyces spp.,Histoplasma spp., Coccidoides spp., Sporothrix spp., Microsporidia spp.,Malassezia spp. and Basidiomycetes.

Typically the infection are caused by Candida sp. such as Candidaalbicans and Candida glabrata.

Biofilm refers to a mode of microbial growth comprising sessile cells,usually within a complex and highly heterogeneous matrix ofextracellular polymers, and characterized by a reduced sensitivity toantifungal agents. Biofilms can contain single species (e.g. afungi/yeast such as C. albicans) or multiple species microorganisms(such as C. albicans, C. glabrata and other microorganisms, preferablyyeasts and/or fungi or even prokaryotes). In a preferred embodiment saidbiofilm is a fungal biofilm, more preferably a Candida species biofilm,comprising one or more of C. albicans, C. glabrata, and/or C. cruse.Biofilms may also comprise or consist of an Aspergillus species (e.g. A.flavus, A. fumigatus, A. clavatus) biofilm or a Fusarium species (e.g.F. oxysporum, F. culmorum). In the context of the present inventionbiofilm typically refers to a Candida albicans biofilm.

The compositions of the present invention generally comprise miconazoleand domiphen bromide in concentrations that provide a synergisticeffect. Synergy occurs when FICI values of 0,5 or less (less than 0,45,or less than 0,4) are encountered. The measurement of FICI values isexplained in the examples section.

The compositions of the present invention can applied as a gels orcream.

Creams are now in the art as oil in water (o/w) or water in oil (w/o)emulsions. They typically contain an emulsifier and a thickener.

Gels are known in the art as transparent preparations containingcellulose ethers or carbomer in water or a water-alcohol mixture.

Treatment in the context of the present invention refers to a completeor partial reduction of yeast and/or a reduction in the symptoms of afungal infection such as itching, burning with urination, and vaginaldischarge.

These formulations allow at the one hand the easy removal of thecomposition from a tube or syringe and on the other hand ensures thatthe composition remains attached at the site of application.

The compositions of the present invention are in general for topical useand formulated to be compatible with the specific site of application(skin, nails, hair). For vulvovaginal applications the composition isformulated as an acidic composition at a pH between 2,75 and 3,5. Theuse of benzoic acid is well known in the art. Other suitable bufferswhich can be used in pharmaceutical compositions to obtain an acidic pHare e.g. HCl and lactic acid.

Since domiphen bromide is highly soluble in water and miconazole and itssalts are insoluble in water, the compositions of the present inventionare preferably a cream with an emulsifying agent, since miconazole andmiconazole nitrate are insoluble in water and domiphen bromide is highlysoluble in water.

Various oil/in water emulsions are known in the art. In the formulationsof the present invention liquid paraffin and lauroyl macrogol-6glycerides are typically used as oily phase.

To stabilise the emulsion emulsifiers such as for example a mixture ofpolyethylene glycol-6, palmitostearate, ethylene glycol stereate andPEG-32 stereate can be used.

To increase the viscosity of the emulsion, thickening agents such ascetostearyl can be used.

For optimal attachment to the site of application formulations furthermay comprise a mucoadhesive. Suitable mucoadhesives for vulvovaginalapplication includes polymers that are capable of forming hydrogels suchas synthetic polycarbophil, chitosan, cellulose derivatives(hydroxyethycellulose, hydroxypropylcellulose andhydroxypropylmethylcellulose), pectin, hyaluronic acid derivatives,polyacrylates, tragacanth, carrageenan and sodium alginate, thiolatedpolymers.

The pharmaceutical applications can be used as a prophylactic, but aretypically used upon signs of a fungal infections. Application of thepharmaceutical composition on the infected region can be performed e.g.every two days, daily or twice three times daily for a period of e.g. atleast 7, 14 or 21 days, or until signs of infection have disappeared.

The compositions of the present invention are equally suitable for thetreatment of recurrent vulvovaginal candidiasis (i.e. at least fourspecific episodes occur in one year or at least three episodes unrelatedto antibiotic therapy occur within one year). The compositions of thepresent invention are suitable for recurrent infection with C. albicansand specifically suitable for recurrent infections with C. tropicalis orC. glabrata, which are less sensitive to miconazole monotherapies.

Pharmaceutical compositions of the present invention comprise betweenabout 1% (w/w) to about 5% (w/w/) miconazole or a salt thereof(typically nitrate). Specific embodiments refer to about 1% (w/w/),about 1,5% (w/w), about 2% (w/w/) about 2,5% (w/w), about 3% (w/w/),about 4%, or about 5% (w/w/) miconazole nitrate.

The amount of miconazole or its salt in the pharmaceutical compositionsof the present is in a molar excess of about 2, about 2,5, about 3,about 3,5 or about 4, over the amount of domiphen bromide.

Thus for example a pharmaceutical composition with a 3 fold molar excesscomprising a 10 millimolar domiphen bromide contains 30 millimolarmiconazole nitrate.

Any combination of the above mentioned concentrations miconazole or saltand above mentioned values of molar excess are disclosed herewith.

Specific embodiments refer to pharmaceutical compositions with about 2%(w/w) miconazole nitrate with a 3 fold molar excess over domiphenbromide.

EXAMPLES Example 1: Materials and Methods

Strains and chemicals. C. albicans strain SC5314 was grown routinely onYPD (1% yeast extract, 2% peptone (International Medical Products,Belgium) and 2% glucose (Sigma-Aldrich, USA)) agar plates at 30° C.Stock solutions of miconazole (MCZ) (Sigma-Aldrich) were prepared inDMSO (VWR International, Belgium). RPMI 1640 medium (pH 7.0) withL-glutamine and without sodium bicarbonate was purchased fromSigma-Aldrich and buffered with MOPS (Sigma-Aldrich). Domiphen bromidewas purchased from Selleck Chemicals and benzalkonium chloride,benzethonium chloride, tetraethylammonium chloride from TCI Europe(Belgium).

Antibiofilm screening assay. A C. albicans SC5314 overnight culture,grown in YPD, was diluted to an optical density of 0.1 (approximately10⁶ cells/mL) in RPMI medium and 100 μL of this suspension was added tothe wells of a round bottomed microplate (TPP Techno Plastic ProductsAG, Switzerland) (30, 31). After 1 h of adhesion at 37° C., the mediumwas aspirated and biofilms were washed with 100 μL phosphate bufferedsaline (PBS) to remove non-adherent cells, followed by addition of 100μL RPMI 1640 medium. Biofilms were allowed to grow for 24 h at 37° C.Afterwards, 10 μM of Miconazole was added in combination with 25 μM of acompound from the Pharmakon 1600 library (2 mM stock solution in DMSO)in RPMI, resulting in 1.1% DMSO background. Biofilms were incubated foran additional 24 h at 37° C. Finally, biofilms were washed andquantified with Cell-Titre Blue (CTB; Promega, USA) (32) by adding 100μL CTB diluted 1/10 in PBS to each well. After 1 h of incubation in thedark at 37° C., fluorescence was measured with a fluorescencespectrometer (Synergy Mx multi-mode microplate reader, BioTek, USA) atλ_(ex) 535 nm and λ_(em) 590 nm. Fluorescence values of the samples werecorrected by subtracting the average fluorescence value of CTB ofuninoculated wells (blank). Percentage of metabolically active biofilmcells was calculated relative to the control treatment (1.1% DMSO).Compounds were considered for retesting when their application in thepresence of 10 μM Miconazole resulted in less than 60% residualmetabolic activity of C. albicans biofilm cells compared to theMiconazole control. This confirmation experiment was performed twice andcompound-only controls were included.

Biofilm checkerboard assay. In order to determine possible synergisticinteractions between antifungal agents on one hand and identifiedpotentiators on the other hand, a checkerboard assay was used. Acombination of antifungal compound (Miconazole, fluconazole, caspofunginor amphotericin B) and potentiator, two-fold diluted across rows andcolumns of a microplate respectively, was added to C. albicans biofilmsgrown as described above (DMSO background 0.6%). After 24 h ofincubation at 37° C., biofilms were quantified with the CTB method asdescribed above. Synergism was determined by FICI (fractional inhibitoryconcentration index) calculations.

The FICI was calculated by the formulaFICI=[C(BEC-2_(A))/BEC-2_(A)]+[C(BEC-2_(B))/BEC-2_(B)], in whichC(BEC-2_(A)) and C(BEC-2_(B)) are the BEC-2 values of the antifungaldrugs in combination, and BEC-2_(A) and BEC-2_(B) are the BEC-2 valuesof antifungal drugs A and B alone. BEC-2 stands for biofilm eradicationconcentration 2 which is the minimal concentration of a compound thatcauses a 2-fold decrease in biofilm metabolic activity.

The interaction was defined as synergistic for a value of FICI≤0.5,indifferent for 0.5<FICI<4 and antagonistic for FICI≥4.

PI assay and CFU determination. Biofilms were grown as described aboveand treated with selected doses of Miconazole and either domiphenbromide, benzalkonium chloride, bezethonium chloride, dequaliniumchloride or tetraethylammonium bromide. To obtain a kinetic PI read out,biofilms were incubated, in the presence of 3% PI, in a multimode reader(Syngergy MX, Biotek) which measures fluorescence each 15 min during 17h (emission wavelength: 535 nm and excitation wavelength: 617 nm). CFUdetermination was performed on biofilms 24 h after treatment. To thisend, biofilms were washed with PBS and thoroughly scraped off the bottomof the plate. Serial dilutions were plated on YPD agar plates andincubated for 48 h at 30° C. before colony counting.

Example 2: Screening for Potentiators of Miconazole Against C. albicansBiofilms

Domiphen bromide (DB), was tested for its capacity to reduce C. albicansbiofilm metabolic activity with more than 50% in combination with asubinhibitory Miconazole dose and its toxicity profile on human cells.

Example 3: Quaternary Ammonium Compounds Act Synergistically withMiconazole Against C. albicans Biofilms

To determine whether domiphen bromide acts synergistically withMiconazole against C. albicans biofilms, checkerboard experiments (FIG.1 ) and FICI calculations were performed (Table 1). The checkerboarddata show that, in the presence of domiphen bromide, lowerconcentrations of Miconazole can be used to reduce the metabolicactivity of C. albicans biofilms, indicative for decreased survival ofthe biofilm cells (FIG. 1 ). Next, we derived the concentration of bothMiconazole and domiphen bromide necessary to eradicate the C. albicansbiofilm with 50% or 2-fold (Biofilm-Eradication-Concentration-2, BEC-2),to enable FICI calculations (Table 1).

TABLE 1 Synergistic Miconazole-domiphen bromide combinations against C.albicans biofilms Concentration DB (μM) BEC-2 MCZ (μM) FICI* 25 1 0.74012.5 30 0.498 ^(##) 6.25 55 0.423 ^(##) 3.125 100 0.527 1.56 150 0.698 0230 Not applicable *FICI calculations are based on the formula FICI =[C(BEC-2A)/BEC-2A] + [C(BEC-2B)/BEC-2B], in which C(BEC-2A) andC(BEC-2B) are the BEC-2 values of Miconazole in combination and BEC-2Aand BEC-2B are the BEC-2 values of Miconazole (230 μM) and domiphenbromide (33 μM) alone. ^(# #) synergistic values

For concentrations of 12.5 μM and 6.25 μM domiphen bromide, synergy wasobserved (FICI<0.5) in combination with Miconazole against C. albicansbiofilms (Table 1). Synergistic combinations were found for combinationsof domiphen bromide and Miconazole in a ratio of 1/9 to 1/2.3.

To our interest, we previously detected other quaternary ammoniumcompounds as potential Miconazole potentiators, namely benzalkoniumchloride (BKC) and bezethonium chloride (BTC) (unpublished data).However, their activity in combination with Miconazole was neverassessed in checkerboard analyses. Therefore, in this study, we assessedpotential synergistic interactions between Miconazole and variousquaternary ammonium compounds, including benzalkonium chloride,bezethonium chloride, dequalinium chloride (DC) and tetraethylammoniumbromide (TB) (FIG. 2 ).

We found that benzalkonium chloride, bezethonium chloride anddequalinium chloride act synergistically with Miconazole (FICI<0.5),while this is not the case for tetraethylammonium bromide (FICI>0.5).These data suggest that the synergistic action with Miconazole is acharacteristic of various quaternary ammonium compounds, implying thatthe activity is probably attributed to the core chemical structure ofthis family.

Example 4: The Domiphen Bromide—Miconazole and BenzalkoniumChloride—Miconazole Combinations Show Prominent Fungicidal ActivityAgainst C. albicans Biofilms

A reduction in metabolic activity, as observed in the previousexperiments, does not necessarily mean that the biofilm cells underinvestigation are dead. It could very well be that their cellularmetabolism is attenuated in the presence of the compounds, but that theyrevive when the stress is removed. To assess how much damage is actuallycaused to the cells and in what time frame, we performed a time-courseexperiment on C. albicans biofilms treated with a combination ofdomiphen bromide, benzalkonium chloride, bezethonium chloride ordequalinium chloride (25 μM or 50 μM) and Miconazole (75 μM or 150 μM)(resulting in a 1/3 ratio) or single compounds, in the presence ofpropidium iodide (PI) (FIG. 3 ). The concentrations of the compoundswere chosen based on the observation in FIG. 1 that 25 μM domiphenbromide in combination with 500, 250, 125 or 62.5 μM Miconazole resultedin a reduction of C. albicans biofilm metabolic activity below 25% (boxin FIG. 1 ).

PI is known to permeate through the membrane of damaged cells and itsuptake is an indication of cell death. We observed only a slightincrease in PI fluorescence when cells were treated with 150 μMMiconazole after 16 h of treatment. Among the single treatments with thequaternary ammonium compounds, only domiphen bromide and benzalkoniumchloride showed a slight increase in PI fluorescence 5-10 h aftertreatment at 50 μM. However, the fastest effect is observed forcombinations of Miconazole with either 50 μM domiphen bromide,benzalkonium chloride or bezethonium chloride, with PI values startingto increase already 2-3 h after treatment. Remarkably, no additionalincrease in PI fluorescence could be observed for combinations ofMiconazole and dequalinium chloride (FIG. 3 ). Remarkably, no increasewas observed for dequalinium chloride, but combinations with 50 μM arelacking.

The killing capacity of the quaternary ammonium compounds in thepresence or absence of Miconazole was further confirmed by CFU countingfor selected doses of the compounds (FIG. 4 ).

Therapeutically interesting reductions in CFUs (>3 Log units) were onlyobserved for combinations of 150 μM Miconazole with 50 μM benzalkoniumchloride and 50 μM domiphen bromide (FIG. 4 ). Interestingly, in a fewcases, complete sterility of the surface was observed after treatmentwith these combinations.

Example 5: The Observed Synergy Between Quaternary Ammonium Compoundsand Miconazole is Miconazole-specific

We assessed whether the quaternary ammonium compound could also increasethe activity of fluconazole against C. albicans biofilms. Fluconazoleshowed no significant antibiofilm activity on its own nor in combinationwith 25 μM domiphen bromide, benzalkonium chloride, bezethonium chlorideor dequalinium chloride (BEC-2>500 μM) (data not shown).

Example 6: Formulations for Vulvovaginal Application

The example illustrates the preparation of different pharmaceuticalcompositions comprising domiphen bromide and/or miconazole.

TABLE 2 formulation of domiphen bromide and/or miconazole compositions.F2 F3 F4 F5 F1 2% MCZ 4% MCZ domiphen 2% MCZ nitrate + 3/1 ExcipiëntPlacebo nitrate nitrate bromide domiphen bromide (property) (w/w %) (w/w%) (w/w %) (w/w %) (w/w %) Mixture of PEG-6 12.00 11.76 11.52 11.9311.69 palmitostearate, ethylene glycol stearate and PEG-32 stearate (O/Wemulsifier) Lauroyl macrogol-6 3.00 2.94 2.88 2.98 2.92 glycerides (Oilyphase) Cetostearyl alcohol 2.00 1.96 1.92 3.98 3.90 (thickener) liquidparaffine 8.00 7.84 7.68 7.95 7.79 (Oily phase) Water (Aqueous 75.0073.50 72.00 72.58 71.12 phase) Miconazole nitrate 0.00 2.00 4.00 0.002.00 Domiphen bromide 0.00 0.00 0.00 0.58 0.58 In F5 miconazole nitrateis in a 3 fold molar excess over domiphen bromide

Example 7: Animal Model for Vaginal Candida Infection

The above F5 formulation is a miconazole nitrate-domiphen bromidepreparation with in vitro synergistic effect (3 fold molar excess ofdomiphen bromide over miconazole nitrate) is tested is a rat model.

F1 to F4 are controls with no active ingredient (F1) miconazole nitrate(F2 and F3) and domiphen bromide (F4)

Female Wistar rats (180-200 g) are kept in groups of 4 animals. Food andwater is available ad libitum. Husbandry conditions are: roomtemperature 22° C., humidity 60% and a day-night cycle of 12 h light/12h dark.

Preconditioning

The rats are ovariectiomized 2 to 3 weeks before infection underinhalation—anesthesia with isoflurane. During recovery, the rats are putunder an IR-lamp for about 30 minutes. Three days before infection, theanimals receive 1 mg oestradiolbenzoate in 0.1 ml PEG400 SC and 200 μgprogesteron in 0.1 ml PBS SC to induce artificial estrus (given as 1injection). This injection is repeated twice during the experiment.

Preparation of Inoculum and Infection Procedure

Candida albicans B2630-cryostock is used for the infection. Each vialcontains 1 ml RPMI with 10% glycerol with an inoculum size of 5.108cfu/ml. A dilution of 1/5 in sterile water is made prior to infection.The animals are infected with 1.107 cfu in 100 μl using a micropipettewith disposable tips. The end of the tip is inserted into the vagina andthe rat is held slightly up by the tail for about one minute afterinstillation.

Treatment Groups

Topical b.i.d. treatment is started on the morning after the infection(D1) for 4 days (200 μl/animal, applied by inserting the tip of a 1 mlsyringe into the vagina).

The following formulations have been used. (details of theseformulations are shown above in table 2).

G1a: Formulation 1a or Vehicle-treated infected control (VICa): b.i.d.(200 μL) (#2)

G1b: Formulation 1b or Vehicle-treated infected control (VICb): b.i.d.(200 μL) (#2)

G2: Formulation 2 (Miconazole nitrate): b.i.d. Top 2% for 4 days (200μL) (#4)

G3: Formulation 3 (Miconazole nitrate): b.i.d. Top 4% for 4 days (200μL) (#4)

G4: Formulation 4 (Domiphen bromide): b.i.d. Top for 4 days (200 μL)(#4)

G5: Formulation 5 (Miconazole nitrate/Domiphen bromide): b.i.d. 4 days(200 μL) (#4)

Evaluation Parameters

Estrus control: on days 4, 9 and 14, vaginal smears are taken with amoist swab and stained with Giemsa to check the artificial estrus(microscopic count of cornified epithelial cells). On days 2 and 7, thehormone injections are repeated in order to maintain a continuousestrus.

Body weight: follow up of body weight (BW) and calculation of percentagegain or loss in comparison to BW before infection (day 0).

Vaginal yeast burdens: vaginal samples are collected on days 4, 9 and14. A sterile swab is moistened in sterile water, inserted into thevagina and rotated twice. The swab is then brought into a tube with 1 mlsterile water and put on the vortex stirrer for 40 seconds. Ten-folddilutions of this solution are prepared and plated on a Sabouraud agar.After 24-48 h incubation at 37° C., the colonies are counted and thenumber of cfu's/ml is calculated as a measure for the total intravaginalburden. The results are expressed as a LOG function of the number ofcfu's that was found on the swab (except when no colonies, where theresult=0).

Results

As there was no differences between both vehicle control groups 1a and1b, both groups were combined and shown as Group 1.

Estrus Control

High levels of cornified epithelial cells were observed throughout theexperiment (Table 3), confirming permanent estrus.

TABLE 3 Percentages of cornified epithelial cells observed throughoutthe experiment on several days p.i. R1 R2 R3 R4 Average G1 Day 0  99%95% 99% 95% 97% Day 4  90% 90% 95% 95% 93% Day 9  90% 80% 95% 90% 89% G2Day 0  99% 95% 95% 99% 97% Day 4  90% 99% 95% 99% 96% Day 9  90% 90% 80%40% 75% G3 Day 0  95% 99% 95% 90% 95% Day 4  99% 99% 90% 95% 96% Day 9 50% 90% 95% 20% 64% G4 Day 0 100% 99% 99% 95% 98% Day 4  90% 90% 95%50% 81% Day 9  50% 95% 85% 95% 81% G5 Day 0  99% 99% 99% 95% 98% Day 4 90% 80% 50% 90% 78% Day 9  90% 90% 60% 95% 84% G: treatment group, R:animal number.

Body Weight

The standard deviation of the average body weight per animal remainedconstant during the course of infection (Table 4), indicating that nosevere weight loss occurred.

TABLE 4 Evolution of live body weight (g) of rats upon vaginal infectionwith Candida albicans day 1 day 2 day 3 day 4 day Average SD (g) (g) (g)(g) 15(g) (g) (g) G1 R1 258 251 254 245 255 253 5 R2 253 247 253 244 248249 4 R3 261 254 253 245 242 251 8 R4 282 275 280 271 276 277 4 G2 R1266 258 256 250 252 256 6 R2 275 272 270 264 264 269 5 R3 264 257 260252 253 257 5 R4 248 243 246 241 239 243 4 G3 R1 276 271 270 265 264 2695 R2 262 254 256 246 254 254 6 R3 264 260 266 256 255 260 5 R4 244 238241 232 235 238 5 G4 R1 252 244 247 238 244 245 5 R2 273 268 272 252 264266 8 R3 261 254 258 251 263 257 5 R4 261 252 256 245 244 252 7 G5 R1240 241 244 235 232 238 5 R2 245 237 243 231 233 238 6 R3 265 255 258251 249 256 6 R4 236 235 237 230 232 234 3 G: treatment group, R: animalnumber, SD: standard deviation.

Vaginal Yeast Burden

The infection is measured by counting the intravaginal fungal burden ofeach swab (FIG. 5 ) and calculating the area under the curve (AUC) (FIG.6 ). This method allows comparison of the total infection of severaltreatment groups to the control group, while cfu counts allow for acomparison at several time points p.i. The AUC clearly shows that 2%miconazole doesn't affect the outcome of the infection, while 4%miconazole has a positive effect (33% reduction in AUC). The potentiatorDOM by itself doesn't influence the outcome of the infection, while thecombination of 2% MIC+DOM reduces AUC of the infection by 66%.

Under the stated experimental conditions, an antifungal effect ofmiconazole when used at 4% could be demonstrated. No antifungal effectwas observed for miconazole at 2%, but a significant effect was seenwhen the potentiator domiphen bromide was added, although domiphenbromide itself does not influence the outcome of the infection.

Example 8: Vaginal Candida Infection (II)

Animals, preconditioning, preparation of inoculum and infectionprocedure is as described in example 7.

Treatment Groups

Topical b.i.d. treatment is started on thee morning after the infection(D1) for 4 days (200 μl/animal, applied by inserting the tip of a 1 mlsyringe into the vagina)

G1: Formulation 1 or Vehicle-treated infected control (VIC): b.i.d. (200μL) (#4)

G2: Formulation 2 (Miconazole nitrate): b.i.d. Top 2% for 4 days (200μL) (#9)

G3: Formulation 5 (Miconazole nitrate/⅓ mmol Domiphen bromide): b.i.d. 4days (200 μL) (#10)

G4: Formulation 4 (⅓ mmol Domiphen bromide): b.i.d. 4 days (200 μL) (#4)

Evaluation Parameters

Estrus control, Body weight and Vaginal yeast burdens were determined asdescribed above.

Estrus Control

High levels of cornified epithelial cells were observed throughout theexperiment (Table 5, which relates to estrus. It should be noted that,for some animals in group 3, cornification seemed to lower around day 4.This is probably due to the way of sampling, which has been modified forday 9, demonstrating cornification is still in place.

TABLE 5 Percentages of cornified epithelial cells observed throughoutthe experiment on several days p.i. G1 R1 R2 R3 R4 Average Day 0 99 100100 100 100 Day 4 99 90 90 90 92 Day 9 80 90 80 99 87 G2 R1 R2 R3 R4 R5R6 R7 R8 R9 Average Day 0 99 100 90 100 100 99 99 100 100 99 Day 4 80 9090 99 90 60 80 99 99 87 Day 9 20 99 95 90 99 0 95 0 99 66 G3 R1 R2 R3 R4R5 R6 R7 R8 R9 R10 Average Day 0 99 100 100 100 100 100 99 95 95 95 98Day 4 80 90 40 50 90 80 60 20 30 50 59 Day 9 100 100 90 90 99 99 100 9099 90 96 G4 R1 R2 R3 R4 Average Day 0 99 99 100 100 100 Day 4 80 90 9060 80 Day 9 90 99 90 90 92 G: treatment group, R: animal number. (allvalues in percentages)

Body Weight

The standard deviation of the average body weight per animal remainedconstant during the course of infection (Table 6), indicating no severeweight loss occurred.

TABLE 6 Body weight (in g) of rats in several days p.i. Day Day Day DayDay Day day Day 2 3 4 7 9 14 Avg. SD 0 (g) 1 (g) (g) (g) (g) (g) (g) (g)(g) (g) G1 238 241 238 232 220 228 232 222 231 8 R1 R2 246 243 243 239229 226 230 228 236 8 R3 259 256 256 247 242 249 252 254 252 6 R4 254255 251 250 240 242 253 245 249 6 G2 257 259 255 254 246 249 254 248 2535 R1 R2 242 246 236 235 226 231 234 232 235 6 R3 245 244 238 237 229 229227 243 237 7 R4 224 224 222 219 213 217 219 216 219 4 R5 259 257 253247 241 260 259 259 254 7 R6 222 222 219 220 212 229 235 238 225 9 R7246 248 241 240 238 238 232 238 240 5 R8 245 244 241 240 230 229 235 229237 7 R9 224 226 223 223 213 215 216 211 219 6 G3 243 235 235 232 224231 239 237 235 6 R1 R2 250 257 253 244 235 239 246 243 246 7 R3 254 254255 246 237 239 246 226 245 10 R4 253 253 250 244 237 237 243 246 245 6R5 266 265 256 255 251 255 262 262 259 5 R6 242 244 239 236 223 218 230226 232 9 R7 263 265 256 256 244 250 258 257 256 7 R8 252 251 246 246238 246 242 253 247 5 R9 212 230 228 227 218 229 235 238 227 8 R10 262260 257 257 246 251 254 250 255 5 G4 246 258 252 250 241 242 246 250 2486 R1 R2 243 249 242 240 232 235 232 236 239 6 R3 233 221 234 229 222 228232 228 228 5 R4 268 268 266 261 256 257 260 268 263 5 G: treatmentgroup, R: animal number, SD: standard deviation.

Vaginal Yeast Burdens

The infection is measured by counting the intravaginal fungal burden ofeach swab (Table 7) and calculating the area under the curve (AUC) (FIG.8 ). This method allows comparison of the total infection of severaltreatment groups to the control group, while cfu counts allow for acomparison at several time points p.i.

The AUC shows a clear effect of miconazole 2%, concurring with previousresults, reducing the infection by 65%. Also concurring with previousresults, the combination of 2% miconazole+potentiator reduces the AUC,but there is only a clear benefit at day 4 when compared to 2%miconazole. Concurring with the first experiment (exp. 2017-1146),domiphen by itself doesn't reduce the infection. For this experiment, noextra agitation of certain groups has been noted, as opposed to previousresults.

TABLE 7 Intravaginal infection burden. G1 1 2 3 4 avg Cfu/ml 1.02 4.4 6.00 3.00 2.39 (D4) E+04 E+04 E+05 E+05 E+05 Log 4.01 4.64 5.78 5.485.38 (D4) Cfu/ml 2.56 1.98 1.56 8.6  5.27 (D9) E+3  E+5  E+3  E+3  E+4 Log 3.41 5.3  3.19 3.93 4.75 (D9) Cfu/ml 2.04 8.2  4.00 3.8  3.80 (D14)E+5  E+2  E+1  E+3  E+4  Log 5.31 0    1.6  3.58 4.72 (D14) G2 1 2 3 4 56 7 8 9 avg Cfu/ml 6.00 0.00 0.00 0.00 6.00 1.00 0.00 4.00 0.00 2.89(D4) E+2  E+2  E+3  E+2  E+2  Log 2.78 0.00 0.00 0.00 2.78 3.00 0.002.60 0.00 2.46 (D4) Cfu/ml 0.00 7.40 0.00 0.00 9.2  0.00 0.00 0.00 0.001.10 (D9) E+4  E+5  E+5  Log 0.00 4.78 0.00 0.00 5.96 0.00 0.00 0.000.00 5.04 (D9) Cfu/ml 0.00 1.18 0.00 0.00 7.4  0.00 0.00 6.60 0.00 8.31(D14) E+3  E+5  E+3  E+4  Log 0.00 3.07 0.00 0.00 5.87 0.00 0.00 3.820.00 4.92 (D14) G3 1 2 3 4 5 6 7 8 9 10 avg Cfu/ml 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 (D4) Log 0.00 0.00 0.00 0.00 0.000.00 0.00 0.00 0.00 0.00 0.00 (D4) Cfu/ml 4.4  0.00 7.20 1.48 8.6  4.801.72 0.00 0.00 1.00 3.82 (D9) E+03 E+04 E+05 E+04 E+02 E+04 E+02 E+04Log 3.64 0.00 4.86 5.17 4.93 2.68 4.24 0.00 0.00 2.00 4.52 (D9) Cfu/ml3.60 0.00 1.66 1.64 1.98 0.00 2.12 1.30 0.00 1.86 7.01 (D14) E+03 E+05E+05 E+05 E+04 E+05 E+04 E+04 Log 3.56 0.00 5.22 5.21 5.30 0.00 4.335.11 0.00 4.27 43.85  (D14) G4 1 2 3 4 avg Cfu/ml 1.22 1.6  7.60 5.003.5  (D4) E+03 E+02 E+03 E+03 E+03 Log (D4) 3.09 2.20 3.88 3.70 3.54Cfu/ml 4.80 4.00 1.42 3.20 1.56 (D9) E+04 E+05 E+05 E+04 E+05 Log (D9)4.68 5.60 5.15 4.51 5.19 Cfu/ml 1.56 5.40 1.18 3.20 1.43 (D14) E+3  E+4 E+3  E+2  E+4  Log (D14) 3.19 4.73 3.07 2.51 4.15

FIG. 7 shows the intravaginal fungal burden of all groups at differentdays p.i. The burden is shown as the log of the colony forming units(cfu) present on the vaginal swab. Errors bars represent SEM. FIG. 8shows Intravaginal fungal burden of all groups. The burden is shown asthe area under the curve (AUC) compared to the control group (G1)(=100%) is shown as the log of the colony forming units (cfu) present onthe vaginal swab. Errors bars represent SEM. G: treatment group.

Both miconazole 2% and miconazole 2% combined with domiphen reduce theinfection. At day 4, there is an additional effect of domiphen, but thiseffect cannot be observed at days 9 and 14.

FIG. 9 summarises the result of 3 experiments obtained with 12 rats ascontrol, 17 rats with treatment with 2% mcz, 8 rats with domiphenbromide only, 18 rats with treatment with 2% MCZ+⅓ DB, 6 rats withtreatment with 2% MCZ+⅙ DB.

Example 9 Potentiation of Miconazole by Domiphen Bromide Against Candidaglabrata Biofilms Materials and Methods

Strains and chemicals. C. glabrata strain BG2 was grown on YPD (1% yeastextract, 2% bacteriological peptone (LabM, UK) and 2% glucose(Sigma-Aldrich, USA)) agar plates at 30° C. Stock solutions ofmiconazole (MCZ) (Sigma-Aldrich) were prepared in DMSO (VWRInternational, Belgium). RPMI 1640 medium (pH 7.0) with L-glutamine andwithout sodium bicarbonate was purchased from Sigma-Aldrich and bufferedwith MOPS (Sigma-Aldrich). Domiphen bromide (DB) was supplied by PurnaPharmaceuticals and a work solution of 1% triton (Sigma-Aldrich) wasprepared in phosphate buffered saline (PBS).

Biofilm checkerboard assay with CFU determination. A C. glabrata BG2overnight culture, grown in YPD at 30° C., was diluted to an opticaldensity of 0.1 (approximately 10⁶ cells/mL) in RPMI medium and 100 μl ofthis suspension was added to the wells of a round bottomed microplate(TPP Techno Plastic Products AG, Switzerland). After 1 h of adhesion at37° C., the medium was aspirated and biofilms were washed with 100 μLPBS to remove non-adherent cells, followed by the addition of 100 μLRPMI 1640 medium. Biofilms were allowed to grow for 24 h at 37° C. Todetermine possible potentiation of MCZ by DB, a checkerboard assay wasused. A combination of MCZ and DB, two fold diluted across rows andcolumns of a microplate respectively, was added to C. glabrata biofilms(DMSO background 1%). After 38 h of incubation at 37° C., CFUdetermination was performed on treated biofilms. To this end, biofilmswere washed with PBS, thoroughly scraped off the bottom of the plate anddissolved in 100 μl triton (1%). Serial dilutions (10⁻², 10⁻³ and 10⁻⁴)were plated on YPD agar plates, followed by an incubation period of 24 hat 37° C. and subsequent colony counting.

Results and Discussion Domiphen Bromide Potentiates Miconazole AgainstC. albicans Biofilms

Quaternary ammonium compounds, like DB, are potentiators of MCZ againstC. albicans biofilms. In order to determine whether DB is also apotentiator of MCZ against C. glabrata biofilms, a checkerboardexperiment was performed FIG. 10 shows the Survival of C. glabratabiofilm cells after single -or combination treatment with MCZ and DB.Mean log CFU values +/−SEM are shown for 6 biological repeats.Statistical analysis was performed to assess significant differencesbetween both single treatments with MCZ (150 μM) and DB (37,5 μM) and acombination. A 2-way ANOVA and Tukey multiple comparisons test wereperformed and significant differences are shown.

Concentration series of both compounds were tested and a significantreduction in CFUs was only observed for a combination of 500 μM MCZ with37,5 μM DB as compared to single compound treatment.

The Observed Potentiation of MCZ by DB Appears to be MCZ-specific

We assessed whether DB could also increase the activity of posaconazoleagainst C. glabrata biofilms. Posaconazole showed no significantantibiofilm activity on its own nor in combination with DB (data notshown).

Example 10 Potentiation of Azoles by Domiphen Bromide Against Candidaalbicans Biofilms

Strains and chemicals. Both C. albicans strain SC5314 and strain B59630,kindly supplied by the laboratory of Microbiology, Parasitology andHygiene from prof. Paul Cos, were grown on YPD (1% yeast extract, 2%bacteriological peptone (LabM, UK) and 2% glucose (Sigma-Aldrich, USA))agar plates at 30° C. Stock solutions of miconazole (MCZ)(Sigma-Aldrich), ketoconazole (TCI Europe, Belgium), clotrimazole(Sigma-Aldrich), itraconazole (TCI) and fluconazole (MP Biomedicals,France) were prepared in DMSO (VWR International, Belgium). Posaconazole(Noxafil) was bought from MSD. RPMI 1640 medium (pH 7.0) withL-glutamine and without sodium bicarbonate was purchased fromSigma-Aldrich and buffered with MOPS (Sigma-Aldrich). Domiphen bromide(DB) was supplied by Purna Pharmaceuticals and a work solution of 1%triton (Sigma-Aldrich) was prepared in phosphate buffered saline (PBS).

Biofilm checkerboard assay with CFU determination. A C. albicansovernight culture, grown in YPD at 30° C., was diluted to an opticaldensity of 0.1 (approximately 10⁶ cells/mL) in RPMI medium and 100 μl ofthis suspension was added to the wells of a round bottomed microplate(TPP Techno Plastic Products AG, Switzerland). After 1 h of adhesion at37° C., the medium was aspirated and biofilms were washed with 100 μLPBS to remove non-adherent cells, followed by the addition of 100 μLRPMI 1640 medium. Biofilms were allowed to grow for 24 h at 37° C. Todetermine possible potentiation of the antifungal agents by DB,checkerboard assays were used. A combination of the antifungal compound,either an imidazole (MCZ, ketoconazole or clotrimazole) or a triazole(fluconazole, itraconazole or posaconazole), and DB, two fold dilutedacross rows and columns of a microplate respectively, was added to C.albicans biofilms. After 24 h of incubation at 37° C., CFU determinationwas performed on treated biofilms. To this end, biofilms were washedwith PBS, thoroughly scraped off the bottom of the plate and dissolvedin 100 μl triton (1%). Serial dilutions (10⁻¹, 10⁻² and 10⁻³) wereplated on YPD agar plates, followed by an incubation period of 24 h at37° C. and subsequent colony counting.

Results and Discussion

Quaternary ammonium compounds, like DB, are potentiators of MCZ againstC. albicans biofilms. Imidazoles, for example MCZ, and triazoles, likefluconazole, are two groups of azoles, characterized by 2-and 3 nitrogenatoms in the azole ring respectively (Cleary et al. (1990). DICP, Annalsof Pharmacother., 24: 148-152; Troyer et al. (2013). J.chem., 2013:1-23). Azoles inhibit ergosterol biosynthesis and miconazole has anadditional effect on the cell's mitochondria (Cleary et al. (1990).DICP, Annals of Pharmacother., 24: 148-152; Ghannoum & Rice (1999).Clinical microbial. reviews, 12(4): 501-517; Portillo & Gancedo (1984)Eur. J Biochem, 143(2): 273-276; Swamy et al. (1974). Antimicrob. AgentsChemother. 5(4): 420-425). In order to determine whether DB alsopotentiates other imidazoles and triazoles against C. albicans biofilms,checkerboard experiments were performed (FIG. 11 ).

FIG. 11 shows survival of C. albicans biofilm cells after single -orcombination treatment with an antimycotic and DB. (panels A-D) Survivalof C. albicans strain B59630 or strain SC5314 biofilm cells after single-or combination treatment with an imidazole and DB. Mean log CFU values+/−SEM are shown for at least 3 biological replicates. Statisticalanalysis was performed to assess significant differences between bothsingle treatments with DB and with either miconazole (A and C) (150 μM),ketoconazole (panel B) (300 μM) or clotrimazole (panel D) (150 μM) andcombinations (DMSO background 1%). A 2-way ANOVA and a Tukey multiplecomparisons test were performed and significant differences are shown.

(E-G) Survival of C. albicans SC5314 biofilm cells after single -orcombination treatments with a triazole and DB. Mean log CFU values+/−SEM are shown for at least 2 biological replicates. Statisticalanalysis was performed to assess significant differences between bothsingle treatments with DB and with either fluconazole (panel E) (150μM), itraconazole (panel F) (75 μM) or posaconazole (panel G) (285 μM)and combinations (DMSO background 1%, 2% and 0,5% respectively). A 2-wayANOVA and a Tukey multiple comparisons test were performed and P-valuesare shown.

Concentration series of both the azole and DB were tested and thecombination, resulting in the most significant CFU reduction, is shownin FIG. 11 . Significant reductions in CFUs were only observed forcombinations of DB with imidazoles as compared to single compoundtreatments. The combination MCZ and DB resulted in significantreductions in CFUs as compared to single compound treatments for both C.albicans strain SC5314 and strain B59630. In a few cases, treatment withthese combinations resulted in complete sterility of the surface.Triazoles showed no significant antibiofilm activity on their own nor incombination with DB, indicating that the observed potentiation ofimidazoles by DB is imidazole-specific.

Example 11. Potentiation of Miconazole by Domiphen Bromide Against C.glabrata Biofilms Materials and Methods

Strains and chemicals. C. glabrata strain BG2 was grown on YPD (1% yeastextract, 2% bacteriological peptone (LabM, UK) and 2% glucose(Sigma-Aldrich, USA)) agar plates at 30° C. Stock solutions ofmiconazole (MCZ) (Sigma-Aldrich) were prepared in DMSO. RPMI 1640 medium(pH 7.0) with L-glutamine and without sodium bicarbonate was purchasedfrom Sigma-Aldrich and buffered with MOPS (Sigma-Aldrich). Domiphenbromide (DB) was supplied by Purna Pharmaceuticals.

Checkerboard assay with determination of the minimal inhibitoryconcentration (MIC). A C. glabrata BG2 overnight culture, grown in YPD,was diluted to an optical density of 0.1 (approximately 10⁶ cells/mL) inRPMI medium, preheated at 37° C. 90 μL of the cell suspension was addedto the wells of a 96 well plate, containing 10 μL of a combination ofMCZ and DB, two fold diluted across rows and columns respectively (DMSObackground 2%). After both 24 h and 48 h of incubation at 37° C.,absorbance was measured at 490 nm by a multimode reader (Synergy Mxmulti-mode microplate reader, BioTek, USA).

Miconazole is Potentiated by DB Against Planktonic C. glabrata Cultures

Domiphen bromide (DB) is a potentiator of miconazole against C. glabratabiofilms. To determine whether DB also potentiates miconazole againstplanktonic C. glabrata cultures, the minimal inhibitory concentration(MIC), the miconazole concentration leading to a reduction in visiblegrowth of 50%, was determined by means of a checkerboard assay (FIG. 12). MIC values were determined using Graphpad Prism 6 (table 8).

FIG. 12 shows that combined MCZ-DB treatment reduces the MIC value ascompared to mono treatment of MCZ against C. glabrata planktoniccultures. The values represent the mean visible growth of 3 biologicalrepeats (A) as a percentage of the control treatment (2% DMSO) and (B)as a percentage of mono treatment with corresponding DB concentration.For different DB concentrations, dose-response curves of miconazole areshown in different colors (0 μM blue; 0,78 μM red; 1,56 μM green and3,125 μM purple). A concentration series of MCZ, ranging from 0,0075 μMto 1 mM was tested. Absorbance was measured after 24 h of treatment.

TABLE 8 MIC values for 24 h treatment with MCZ-DB combination againstplanktonic C. glabrata cultures. [DB] (μM) 0 0.78 1.56 3.125 MIC value(μM) MCZ + DB (24 h) 6.772277 7.227962 6.815345 0.1943 (# #) MIC values,significantly reduced as compared to the mono treatment with MCZ, areindicated in green by # #. Graphpad Prism 6 was used to calculate MICvalues.

The checkerboard data show that, in the presence of DB, significantlylower concentrations of MCZ can be used to reduce visible growth with50% as compared to mono treatment with miconazole. A significantreduction of the MIC value was observed for 3,125 μM DB.

Furthermore, benzalkonium chloride (BKC), another quaternary ammoniumcompound, could not potentiate MCZ against planktonic C. glabratacultures (See FIG. 12C).

1. A method for treating of preventing a fungal infection in an animalin need thereof, the method comprising topically administering aneffective amount of a composition comprising an imidazole or a saltthereof, and domiphen bromide as active ingredients wherein saidimidazole is chosen from ketoconazole and clotrimazole.
 2. The methodaccording to claim 1, wherein the fungal infection is an infection withCandida sp.
 3. The method according to claim 1, wherein the fungalinfection is an infection with Candida albicans.
 4. The method accordingto claim 1, wherein the fungal infection is a biofilm formed by Candidaalbicans.
 5. The method according to claim 1, wherein the fungalinfection is a vulvovaginal infection.
 6. The method according to claim1, wherein the fungal infection is recurrent vulvovaginal candidiasis.7. The method according to claim 1, wherein said imidazole or saltthereof and said domiphen bromide are present in a synergisticallyeffective concentration.
 8. The method according to claim 1, whereinsaid imidazole or salt thereof is present in a molar excess of 6 overdomiphen bromide.